Vaccine Research and Development Center, National Health Research Institutes, Zhunan, Taiwan Authority.
PLoS One. 2011 Jan 24;6(1):e14578. doi: 10.1371/journal.pone.0014578.
Highly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system.
The current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3 × 10(6) cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1 ± 0.3 × 10(8) pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera.
The advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.
高致病性流感病毒构成持续威胁，可能导致全球大流行。疫苗接种仍然是减少此类大流行发病率和死亡率的主要措施。在准备计划中需要解决大流行性疫苗的供应和需求激增问题。本研究提出了一种改良的高产量流感 H5N1 疫苗生产工艺，使用无血清（SF）培养基微载体细胞培养系统培养 Madin-Darby 犬肾（MDCK）细胞。
本研究评估了从含血清（SC）培养基切换到几种商业 SF 培养基的细胞适应性。选择的 SF 培养基进一步在各种生物反应器培养系统中进行了工艺放大评估。在所研究的不同体积的生物反应器中，细胞生长没有发现显著差异。在 7.5 L 生物反应器运行中，细胞浓度在 5 天后达到 2.3×106 个细胞/mL。在感染 3 天后，获得了 1024 血凝素（HA）单位/50 µL 和 7.1±0.3×108 pfu/mL 的最大病毒滴度。通过 SRID 测定的 HA 抗原浓度被发现为 14.1 µg/mL，高于从 SC 培养基获得的值。小鼠免疫原性研究表明，用铝佐剂配制的福尔马林灭活的 SF 疫苗候选物能够诱导与从 SC 培养基获得的相似的病毒中和抗体滴度的保护水平。此外，从 SC 或 SF 培养基生产的 H5N1 病毒与 NIBRG14 标准抗血清具有相同的抗原反应性。
这种基于 SF 细胞的生产工艺的优点可以减少动物血清污染、降低 SC 培养基生产成本和批次间的变化。本研究为计划使用 SF 培养基进行细胞基流感疫苗生产的制造商提供了有用的信息。
